【摘要】： Gˉ桿菌感染過程中,其產(chǎn)生的內(nèi)毒素引起的感染性休克和多器官功能障礙綜合癥(multiple organs dysfunction syndrome, MODS)被認(rèn)為是感染及內(nèi)毒素血癥患者死亡的根本原因。細(xì)菌內(nèi)毒素又稱為脂多糖(lipopolysaccharide, LPS),是該類病癥的主要致病物質(zhì)。脂多糖結(jié)合蛋白(Lipopolysaccharide Binding Protein,LBP)是一種促進(jìn)LPS與效應(yīng)細(xì)胞受體相結(jié)合的關(guān)鍵載體蛋白,在炎癥反應(yīng)過程中具有重要的作用。 研究表明,LBP能顯著促進(jìn)LPS與單核/巨噬細(xì)胞膜表面的CD14(membraneCD14 mCD14)結(jié)合,形成LPS-LBP-mCD14復(fù)合物,經(jīng)TLR4-MD-MYD88和NF-Kappa B途徑,激活單核/巨噬細(xì)胞,促進(jìn)炎癥的發(fā)生、發(fā)展;LBP的N、C端分別為與LPS和mCD14的結(jié)合區(qū),因此若能研究獲得針對LBP的抗體,特別是針對LBP N端(N-teminatal fragment of human LBP,NH-LBP)的抗體,則可能阻斷LPS與LBP的結(jié)合,從而降低內(nèi)毒素介導(dǎo)的炎癥反應(yīng),降低Gˉ桿菌感染及膿毒性休克的死亡率。 本研究以人LBP及人NH-LBP為抗原,對本課題組構(gòu)建的人源噬菌體抗體庫(Fab)進(jìn)行篩選,獲得針對人源LBP及人源NH-LBP均有高結(jié)合力的單克隆抗體菌株;通過mega-PCR引入半胱氨酸(Cys)及酶切位點(diǎn),并連接表達(dá)載體pET-28a(+),使得dsFvV_H鏈和dsFvV_L鏈分別在工程菌BL21 star(DE3)中高效表達(dá)。抽提其包涵體并純化,隨后使dsFvV_H鏈和dsFvV_L鏈蛋白在體外復(fù)性,通過半胱氨酸形成二硫鍵(-S-S-)連接,獲得完整的針對LBP特別是NH-LBP的二硫鍵穩(wěn)定Fv單克隆抗體(disulfide stabilized Fv fragment antibody dsFv),并初步研究其體內(nèi)外生物學(xué)活性。 本研究的主要結(jié)果和結(jié)論如下: 1、以人LBP為抗原,以人源噬菌體抗體庫(Fab)進(jìn)行5輪篩選,在篩選過程中使抗體效價由3.8×10~8升高為3.1×10~(13),提高倍數(shù)為8.15×10~4倍,證明篩選效果良好。 2、獲得的抗體庫質(zhì)粒(pComb3H)切除geneⅢ后,以LBP及NH-LBP為抗原經(jīng)ELISA法鑒定,獲得編碼與人LBP及NH-LBP皆有高結(jié)合力的單克隆抗體菌株(4號菌),其表達(dá)抗體的結(jié)合力對于LBP為陰性對照的4.2倍,對于NH-LPB為陰性對照的3.8倍;通過對4號菌的質(zhì)粒序序列測定及其表達(dá)上清的Western-Blotting檢測,確定其表達(dá)的可溶性抗體Fab與LBP和NH-LBP可良好結(jié)合。
[Abstract]:Endotoxin-induced septic shock and multiple organ dysfunction syndrome (multiple organs dysfunction syndrome, MODS) are considered to be the root causes of death in patients with infection and endotoxemia. Bacterial endotoxin, also known as lipopolysaccharide (lipopolysaccharide, LPS), is the main pathogenic substance of this disease. Lipopolysaccharide binding protein (Lipopolysaccharide Binding Protein,LBP) is a key carrier protein that promotes the binding of LPS to effector cell receptors and plays an important role in inflammatory reaction. It has been shown that LBP can significantly promote the binding of LPS to CD14 (membraneCD14 mCD14) on the surface of mononuclear / macrophage cell membrane and form LPS-LBP-mCD14 complex. Through TLR4-MD-MYD88 and NF-Kappa B pathway, mononuclear / macrophage can be activated and inflammation can be promoted. Development; The C terminal of LBP is the binding region of LPS and mCD14, so if we can obtain the antibody against LBP, especially against LBP N terminal (N-teminatal fragment of human LBP,NH-LBP), we may block the binding of LPS to LBP. Thus, the inflammatory response mediated by endotoxin was reduced, and the mortality of infection and septic shock was decreased. In this study, human LBP and human NH-LBP were used as antigens to screen the human phage antibody library (Fab) constructed by our team, and the monoclonal antibody strains with high binding ability to both human LBP and human NH-LBP were obtained. Cysteine (Cys) and endonuclease sites were introduced by mega-PCR and ligated with expression vector pET-28a (), to make dsFvV_H and dsFvV_L chains highly expressed in BL21 star (DE3). The inclusion bodies were extracted and purified, then the dsFvV_H and dsFvV_L chain proteins were renatured in vitro, and disulfide bonds (-S-) were formed by cysteine. The complete disulfide bond of LBP, especially NH-LBP, was obtained to stabilize the Fv monoclonal antibody (disulfide stabilized Fv fragment antibody dsFv), and its biological activity in vitro and in vivo was preliminarily studied. The main results and conclusions of this study are as follows: 1. Using human LBP as antigen and human phage antibody library (Fab) for 5 rounds, the titer of antibody was increased from 3.8 脳 10 ~ (8) to 3.1 脳 10 ~ (13). The improvement was 8.15 脳 10 ~ (-1) times, which proved that the screening effect was good. 2. The obtained antibody library plasmid (pComb3H) was excised from gene 鈪，

本文編號：2372855